Chen, C. C., et al. (2015). Establishment of centromeric chromatin by the CENP-A assembly factor CAL1 requires FACT-mediated transcription. Dev Cell 34: 73-84. PubMed ID: 26151904Summary:
Centromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. This study uncovered a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, CENP-A assembly by its loading factor, CAL1, was shown to require RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. This work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation.

Beck, E. A. and Llopart, A. (2015). Widespread positive selection drives differentiation of centromeric proteins in the Drosophila melanogaster subgroup. Sci Rep 5: 17197. PubMed ID: 26603658Summary:
Rapid evolution of centromeric satellite repeats is thought to cause compensatory amino acid evolution in interacting centromere-associated kinetochore proteins. Cid, a protein that mediates kinetochore/centromere interactions, displays particularly high amino acid turnover. Rapid evolution of both Cid and centromeric satellite repeats led to a hypothesis that the apparent compensatory evolution may extend to interacting partners in the Condensin I complex (i.e., SMC2, SMC4, Cap-H, Cap-D2, and Cap-G) and HP1s. Missense mutations in these proteins often result in improper centromere formation and aberrant chromosome segregation, thus selection for maintained function and coevolution among proteins of the complex is likely strong. This study reports evidence of rapid evolution and recurrent positive selection in seven centromere-associated proteins in species of the Drosophila melanogaster subgroup, and further postulate that positive selection on these proteins could be a result of centromere drive and compensatory changes, with kinetochore proteins competing for optimal spindle attachment.

Boltengagen, M., Huang, A., Boltengagen, A., Trixl, L., Lindner, H., Kremser, L., Offterdinger, M. and Lusser, A. (2015). A novel role for the histone acetyltransferase Hat1 in the CENP-A/CID assembly pathway in Drosophila melanogaster. Nucleic Acids Res [Epub ahead of print]. PubMed ID: 26586808Summary:
The incorporation of CENP-A (Centromere identifier) into centromeric chromatin is an essential prerequisite for kinetochore formation. Yet, the molecular mechanisms governing this process are surprisingly divergent in different organisms. While CENP-A loading mechanisms have been studied in some detail in mammals, there are still large gaps to understanding of CENP-A/Cid loading pathways in Drosophila. This study reports on the characterization and delineation of at least three different CENP-A preloading complexes in Drosophila. Two complexes contain the CENP-A chaperones CAL1, FACT (subunit Dre4) and/or Caf1/Rbap48. Notably, a novel complex was identified consisting of the histone acetyltransferase Hat1, Caf1 and CENP-A/H4. Hat1 is required for proper CENP-A loading into chromatin, since knock-down in S2 cells leads to reduced incorporation of newly synthesized CENP-A. In addition,CENP-A/Cid interacts with the HAT1 complex via an N-terminal region, which is acetylated in cytoplasmic but not in nuclear CENP-A. Since Hat1 is not responsible for acetylation of CENP-A/Cid, these results suggest a histone acetyltransferase activity-independent escort function for Hat1. Thus, these results point toward intriguing analogies between the complex processing pathways of newly synthesized CENP-A and canonical histones.

Rosin, L. and Mellone, B.G. (2016).Co-evolving CENP-A and CAL1 domains mediate centromeric CENP-A deposition across Drosophila species. Dev Cell 37: 136-147. PubMed ID: 27093083Summary:Centromeres mediate the conserved process of chromosome segregation, yet centromeric DNA and the centromeric histone, CENP-A, are rapidly evolving. The rapid evolution of Drosophila CENP-A loop 1 (L1) is thought to modulate the DNA-binding preferences of CENP-A to counteract centromere drive, the preferential transmission of chromosomes with expanded centromeric satellites. Consistent with this model, CENP-A from Drosophila bipectinata (bip) cannot localize to Drosophila melanogaster (mel) centromeres. It was shown that this result is due to the inability of the mel CENP-A chaperone, CAL1, to deposit bip CENP-A into chromatin. Co-expression of bip CENP-A and bip CAL1 in mel cells restores centromeric localization, and similar findings apply to other Drosophila species. Two co-evolving regions, CENP-A L1 and the CAL1 N terminus, were identified as critical for lineage-specific CENP-A incorporation. Collectively, these data show that the rapid evolution of L1 modulates CAL1-mediated CENP-A assembly, suggesting an alternative mechanism for the suppression of centromere drive.

Garcia Del Arco, A., Edgar, B. A. and Erhardt, S. (2018). In vivo analysis of centromeric proteins reveals a stem cell-specific asymmetry and an essential role in differentiated, non-proliferating cells. Cell Rep 22(8): 1982-1993. PubMed ID: 29466727Summary:
Stem cells of the Drosophila midgut (ISCs) are the only mitotically dividing cells of the epithelium and, therefore, presumably the only epithelial cells that require functional kinetochores for microtubule spindle attachment during mitosis. The histone variant CENP-A marks centromeric chromatin as the site of kinetochore formation and spindle attachment during mitotic chromosome segregation. This study shows that centromeric proteins distribute asymmetrically during ISC division. Whereas newly synthesized CENP-A is enriched in differentiating progeny, CENP-C is undetectable in these cells. Remarkably, CENP-A persists in ISCs for weeks without being replaced, consistent with it being an epigenetic mark responsible for maintaining stem cell properties. Furthermore, CENP-A and its loading factor CAL1 were found to be essential for post-mitotic, differentiating cells; removal of any of these factors interferes with endoreduplication. Taken together, it is proposed two additional roles of CENP-A: to maintain stem cell-unique properties and to regulate post-mitotic cells.

Bobkov, G. O. M., Gilbert, N. and Heun, P. (2018). Centromere transcription allows CENP-A to transit from chromatin association to stable incorporation. J Cell Biol [Epub ahead of print]. PubMed ID: 29626011Summary:Centromeres are essential for chromosome segregation and are specified epigenetically by the presence of the histone H3 variant CENP-A. In flies and humans, replenishment of the centromeric mark is uncoupled from DNA replication and requires the removal of H3 "placeholder" nucleosomes. Although transcription at centromeres has been previously linked to the loading of new CENP-A, the underlying molecular mechanism remains poorly understood. This study used Drosophila melanogaster tissue culture cells to show that centromeric presence of actively transcribing RNA polymerase II temporally coincides with de novo deposition of dCENP-A. Using a newly developed dCENP-A loading system that is independent of acute transcription, it was found that short inhibition of transcription impaired dCENP-A incorporation into chromatin. Interestingly, initial targeting of dCENP-A to centromeres was unaffected, revealing two stability states of newly loaded dCENP-A: a salt-sensitive association with the centromere and a salt-resistant chromatin-incorporated form. This suggests that transcription-mediated chromatin remodeling is required for the transition of dCENP-A to fully incorporated nucleosomes at the centromere.

Teixeira, J. R., Dias, G. B., Svartman, M., Ruiz, A. and Kuhn, G. C. S. (2018). Concurrent duplication of Drosophila Cid and Cenp-C genes resulted in accelerated evolution and male germline-biased expression of the new copies. J Mol Evol. PubMed ID: 29934734Summary:
Despite their essential role in the process of chromosome segregation in eukaryotes, kinetochore proteins are highly diverse across species, being lost, duplicated, created, or diversified during evolution. Surprisingly, the Drosophila CenH3 homolog Cid underwent four independent duplication events during evolution. Particularly interesting are the highly diverged Cid1 and Cid5 paralogs of the Drosophila subgenus, which are probably present in over one thousand species. Given that CenH3 and Cenp-C likely co-evolve as a functional unit, this study investigated the molecular evolution of Cenp-C in species of Drosophila. Yet another Cid duplication (leading to Cid6) was found within the Drosophila subgenus; and not only Cid, but also Cenp-C is duplicated in the entire subgenus. The Cenp-C paralogs, which were named Cenp-C1 and Cenp-C2, are highly divergent. Both Cenp-C1 and Cenp-C2 retain key motifs involved in centromere localization and function, while some functional motifs are conserved in an alternate manner between the paralogs. Interestingly, both Cid5 and Cenp-C2 are male germline-biased and evolved adaptively. However, it is currently unclear if the paralogs subfunctionalized or if the new copies acquired a new function. These findings point towards a specific inner kinetochore composition in a specific context (i.e., spermatogenesis), which could prove valuable for the understanding of how the extensive kinetochore diversity is related to essential cellular functions.

Moreno-Moreno, O., Torras-Llort, M. and Azorin, F. (2019). The E3-ligases SCFPpa and APC/CCdh1 co-operate to regulate CENP-ACID expression across the cell cycle. Nucleic Acids Res. PubMed ID: 30753559Summary:Centromere identity is determined by the specific deposition of CENP-A, a histone H3 variant localizing exclusively at centromeres. Increased CENP-A expression, which is a frequent event in cancer, causes mislocalization, ectopic kinetochore assembly and genomic instability. Proteolysis regulates CENP-A expression and prevents its misincorporation across chromatin. How proteolysis restricts CENP-A localization to centromeres is not well understood. ^hie e6ury reports that, in Drosophila, CENP-ACID expression levels are regulated throughout the cell cycle by the combined action of SCFPpa and APC/CCdh1. SCFPpa regulates CENP-ACID expression in G1 and, importantly, in S-phase preventing its promiscuous incorporation across chromatin during replication. In G1, CENP-ACID expression is also regulated by APC/CCdh1. This study also shows that Cal1, the specific chaperone that deposits CENP-ACID at centromeres, protects CENP-ACID from SCFPpa-mediated degradation but not from APC/CCdh1-mediated degradation. These results suggest that, whereas SCFPpa targets the fraction of CENP-ACID that is not in complex with Cal1, APC/CCdh1 mediates also degradation of the Cal1-CENP-ACID complex and, thus, likely contributes to the regulation of centromeric CENP-Asup>CID deposition.

Centromeres are the chromosomal regions responsible for poleward movement at meiosis and mitosis, and are essential for the faithful segregation of
genetic information. Centromeres of most organisms are embedded within constitutive heterochromatin, the condensed regions of chromosomes that account for a large fraction of complex genomes. Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Centromeric chromatin is distinguished from bulk chromatin, most
conspicuously by the presence of centromere-specific histone H3 variants (CenH3). A Drosophila CenH3, Cid (for
Centromere identifier), the Drosophila homolog of the CENP-A centromere-specific H3-like proteins, localizes exclusively to fly centromeres. The function of Cid is highly conserved. This chromatin component probably plays a key role in
assembling the kinetochore at meiosis and mitosis. Thus, CenH3 could be
expected to both interact with the underlying centromeric DNA, as well
as interact with other proteins (including other CenH3 molecules) to
provide the foundation for the kinetochore (Malik, 2002).

Studies show that centromeres and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that Cid is required for
normal kinetochore formation and function, as well as cell-cycle progression.
Injection of Cid antibodies into
early embryos, as well as RNA interference in tissue-culture cells, shows that Cid is required for several mitotic processes. Cid chromatin is physically separate from proteins involved in sister cohesion (Mei-s332), centric condensation (Prod, kinetochore function (Rough deal, Zeste-white 10 and Bub1) and heterochromatin structure (HP1). Cid localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of Polo, the kinesin kinetocore motor CENP-meta, Rough deal (Rod), Bub1 and Mei-S332, involved in sister chromatid cohesion) depends on the presence of functional Cid. It is concluded that Cid directs kinetochore formation and function by forming a unique heterochromatin within the centromere (Blower, 2001).

Immunolocalization experiments in mammals have shown that it is possible to distinguish between the inner and outer kinetochore by fluorescence microscope. To determine whether Cid is located in the inner kinetochore, Cid, spindle microtubules and many transient kinetochore proteins including Bub1, Zeste-white 10

(ZW10 -- accepted FlyBase name: Mitotic 15) and Rod were simultaneously localized. Each of these proteins would be expected to localize to the outer kinetochore plate or the fibrous corona, similar to other transient kinetochore proteins localized in mammals (for example, Bub1, CENP-E, Dynein) (Blower, 2001).

Simultaneous detection of Cid with outer kinetochore proteins showed that Cid is consistently separated from ZW10, Rod and Polo kinase, and is located closer to the chromosome and further from the kinetochore microtubules than these proteins. Cid is also offset from Bub1 (a component of the spindle assembly checkpoint) at unattached kinetochores, but Cid and Bub1 show significant overlap. This result is consistent with studies in mammals, which suggest that Bub1 may be located at both the inner and outer kinetochore plates. These results show that Cid is located in or near the inner plate of the kinetochore in Drosophila and is likely to be associated closely with centromeric DNA (Blower, 2001).

Previous work has shown that the outer kinetochore proteins ZW10 and Dynein are present on fully functional Drosophila minichromosomes (that is, 100% transmission through mitosis and meiosis), as well as structurally acentric minichromosomes that lack detectable centromeric sequence (neocentromeres). To determine the relationship of Cid-containing chromatin to the functional centromere, the localization of Cid protein was examined in a series of minichromosome derivatives of decreasing size and meiotic transmission efficiency (Blower, 2001).

Cid is present on all minichromosome derivatives that contain a fully functional centromere (gamma238, 31E, 10B and J21A, indicating that Cid colocalizes with the molecular-genetically defined centromere. Cid also is present on all of the neocentromeric derivatives that lack centric heterochromatin, including the normal minichromosome centromere (26C, J19B), consistent with the
localization pattern of outer kinetochore components ZW10 and Dynein. It is concluded that Cid localization is correlated with centromere function, regardless of the composition of the underlying DNA (Blower, 2001).

The presence of Cid at the functional minichromosome centromere shows that Cid localization is correlated with centromere activity. To test the role of Cid in mitosis directly, affinity-purified chicken anti-Cid antibodies were injected into early embryos that express histone H2A/green fluorescent protein (GFP) and chromosome behavior was observed using time-lapse microscopy. Rhodamine-labelled anti-Cid antibodies bind specifically to centromeres in all stages of the cell cycle, and show no nonspecific crossreactivity with histone H3 either in vivo or by Western blot. Injected antibody binds centromeres in a gradient in which more antibody binds close to the site of injection (Blower, 2001).

Injection of Cid antibodies into early embryos results in a range of phenotypes affecting both cell-cycle progression and mitotic chromosome segregation. The phenotypic series is consistent with a gradient of Cid inhibition mirroring the gradient of antibody concentration. Nuclei closest to the site of injection arrest in interphase (13%), whereas nuclei further from the site of injection delayed entering mitosis and exhibited different mitotic defects: specifically, entry into prophase condensation followed by a loss of condensation (3.6%; metaphase arrest [15%]); and various anaphase chromosome segregation defects (failure to move toward the poles at anaphase onset, unequal chromosome segregation, failure to maintain spindle contact and karyokinesis defects at telophase, 20%) (Blower, 2001).

Cid function in Drosophila Kc tissue-culture cells is also disrupted using RNAi. Cells were treated with dsRNA corresponding to the whole Cid transcript; cells were then fixed and monitored for levels of Cid protein and aberrant chromosome behaviour every 24 h after adding dsRNA. Cells in a given treated population display a variable penetrance of Cid inhibition, which results in different phenotypes (Blower, 2001).

Mitotic defects in Kc cells are consistent with those observed after antibody injection into embryos, including aberrant prometaphase congression, precocious sister chromatid separation, kinetochore microtubule capture and anaphase segregation. Cells treated with dsRNA were no longer dividing 8-10 d after RNAi, suggesting that interphase arrest results from complete Cid disruption; however, without live analysis it is difficult to differentiate between interphase arrest and a terminal phenotype resulting from massive chromosome segregation defects (Blower, 2001).

The results of Cid disruption by both antibody injection into embryos and RNAi in tissue-culture cells show that Cid is directly or indirectly required for many aspects of kinetochore-mediated chromosome movement as well as cell-cycle progression (Blower, 2001).

Centromeres in most higher eukaryotes are embedded in centric heterochromatin, suggesting that both the structure and function of heterochromatin are required for centromere function. What are the structural relationships between centromeric chromatin, defined by Cid, and chromosomal proteins previously localized to the centromere region? This question was addressed using immunolocalization of three proteins and Cid on mitotic chromosomes from S2 and Kc tissue-culture cells (Blower, 2001).

Mei-S332 is required for sister chromatid cohesion during metaphase I of meiosis, and is present in the centromeric regions of meiotic and mitotic chromosomes. Simultaneous localization of Cid with Mei-S332 shows that Cid antibodies yield typical double-dot staining, whereas Mei-S332 is localized in two concentrated foci joined by a bridge of staining that connects the sister chromatids. Although Mei-S332 has been described as centromeric and possibly located to the inner kinetochore, the higher resolution localization of Mei-S332 presented in this study showed consistent offset of antibody staining to one side of the kinetochore and along the chromosome axis on all chromosomes. The offset localization is always to the same side of the kinetochore on each chromosome type. This is especially evident on the X chromosome, in which Mei-S332 is always located on the proximal long arm side of Cid, and for chromosomes 2 and 3, on the basis of colocalization with the sequence-specific satellite binding protein Prod. It is concluded that Mei-S332 is located near but not in the Cid chromatin, providing a physical basis for the previous observation that kinetochore function and Mei-S332-mediated cohesion can be separated using minichromosome derivatives (Blower, 2001).

proliferation disrupter (prod) mutant larval neuroblasts display hypo-condensation of the centromere region and metaphase/anaphase arrest. Consistent with the decondensation phenotype, the Prod protein localizes to the centromeric region of chromosomes 2 and 3 in mitosis, suggesting that it may be involved in kinetochore function on these chromosomes. However, simultaneous detection of Cid and Prod on mitotic chromosomes shows that Prod stains a more expansive portion of the chromosome than Cid, and is offset from the kinetochore in the same manner as Mei-S332. In fact, Prod and Mei-S332 are both localized to the same side of the kinetochore on chromosomes 2 and 3 (Blower, 2001).

HP1 mutants show dominant suppression of heterochromatin-induced position-effect variegation (PEV), and recessive telomere fusions and chromosome segregation defects. Human and mouse homologs of HP1 localize to the centromere region, and S. pombe Swi6, another chromodomain protein, is localized to the centromere and required for proper chromosome transmission. Simultaneous localization of Cid with HP1 revealed that HP1 is not present in centromeric chromatin in either interphase or metaphase. In metaphase chromosomes, HP1 is located throughout the pericentric heterochromatin, and is near but not in Cid chromatin (Blower, 2001).

It is concluded that Prod and HP1 are located in the pericentric heterochromatin and not in the centromeric chromatin. These results suggest that, although the centromere is embedded in large blocks of heterochromatin, centromeric chromatin is spatially separable from canonical centric heterochromatin (Blower, 2001).

Although Cid localization is not dependent on the presence of Prod, HP1, Mei-S332 or Polo kinase, the mutant analyses did not determine whether the localization of these proteins depended on Cid. Therefore, Polo kinase, Mei-S332 and Prod localization were examined in embryos in which Cid function was inhibited. In embryonic nuclei close to the site of injection, where high levels of Cid antibody binding and the most severe mitotic defects are observed, Polo kinase localization is diffuse and apparently absent from kinetochores, as judged by counterstaining with Prod. Notably, in these same nuclei Mei-S332 was absent from the pericentromeric region, whereas Prod, a protein with sequence-specific satellite-binding properties, was still present in the pericentromeric region (Blower, 2001).

The localizations of Rod, CENP-meta outer kinetochore, CENP-E kinesin-like protein homolog, Polo kinase, Bub1 and Mei-S332, but not Prod or HP1, were also disrupted in Kc cells displaying mitotic defects as a result of RNAi inhibition of Cid expression. Quantitative deconvolution microscopy has revealed that transient kinetochore component recruitment is proportional to the amount of Cid present at the kinetochore, whereas Prod recruitment is independent of Cid levels. Thus, Cid function is required for the recruitment or maintenance of transient kinetochore components and a centric cohesion protein (Mei-S332), but is not required for the localization of Prod or HP1. These results also indicate that the pleiotropic mitotic defects observed in anti-Cid injection and RNAi are likely to be caused by a failure to recruit or bind transient kinetochore components and a centric cohesion protein (Blower, 2001).

CENP-A has been proposed to be an epigenetic mark required for determining centromere identity and thus the assembly of the kinetochore. This study has shown that Cid, the Drosophila CENP-A homolog, is associated with the inner kinetochore and that Cid localization correlates with centromere function, specifically the presence of the molecular-genetically defined centromere and neocentromere DNA (Blower, 2001).

The presence of Cid on neocentromeres shows that these structurally acentric yet functional chromosomes have acquired centromeric chromatin, consistent with the presence of outer kinetochore components. Furthermore, the presence of Cid on neocentromeres shows that the location of centromeric chromatin is independent of sequence, and that centromeric chromatin can confer centromere identity on normally non-centromeric DNA -- a state that is then propagated faithfully through replication and division (Blower, 2001).

Cid is required for kinetochore assembly and cell-cycle progression in early Drosophila embryos and Kc tissue-culture cells. Mitotic defects have been observed in human cells after CENP-A antibody injection and in the mouse CENP-A knockout; however, the 'live studies' reported in this study have allowed an examination of the temporal and cytological effects of CENP-A/Cid disruption in greater detail. Both antibody injection into embryos and RNAi inhibition in Kc cells results in several defects expected for centromere dysfunction: specifically, aberrant prometaphase congression; chromosome attachment to spindle microtubules; entry into anaphase; anaphase poleward segregation,and failure to resolve properly at telophase (Blower, 2001).

The mislocalization of Rod, Bub1, CENP-meta, Polo and Mei-S332 in nuclei displaying missegregation phenotypes shows that the defects are correlated with aberrant kinetochore structure and the recruitment of transient kinetochore proteins and other centromere region proteins. These results extend the earlier observation that the inner kinetochore protein CENP-C is mislocalized in the CENP-A knockout mouse to the location of outer kinetochore proteins. Notably, the amount of outer kinetochore components present at the kinetochore is proportional to the amount of Cid, suggesting that the kinetochore may be composed of a repeated substructure (Blower, 2001).

Cid disruption may decrease the number or size of functional subunits; this is sufficient to cause defects in mitosis because mitotic defects are observed in cells with decreased but visible amounts of Cid. Disruption of the centromere/kinetochore substructure may be responsible for different degrees of mislocalization of outer kinetochore components, which results in the observed pleiotropy of mitotic phenotypes. Karyokinesis defects observed after Cid inhibition may be the result of the failure of chromosomal passenger proteins to localize to the kinetochore and consequently to the spindle and midbody. On the basis of the mislocalization of several outer kinetochore components, it is concluded that Cid is epistatic to transient kinetochore components; these results support the hypothesis that CENP-A proteins are involved directly in the epigenetic marking of the site of kinetochore formation, and show conclusively that proper kinetochore function is required for many cell-division processes (Blower, 2001).

More complete Cid disruption in embryos results in a severe interphase arrest phenotype, showing that Cid function is also required before entry into mitosis, which is similar to one of the phenotypes observed after injection of anti-CENP-A antibodies into HeLa cells. The use of real-time analysis allows for an unambiguous conclusion that the nuclei are arrested before entry into mitosis. The interphase arrest phenotype suggests that Cid functions in interphase, where it is constitutively bound to centromeres, and that there may be another cell-cycle checkpoint that monitors kinetochore assembly and blocks entry into mitosis if this process is compromised (Blower, 2001).

This putative 'kinetochore assembly' checkpoint is also likely to be responsible for the delay in entering mitosis observed in nuclei just distal to the injection site. It is possible that disruption of an interaction between Polo kinase and Cid is responsible for this cell-cycle arrest because Polo is required for entry into mitosis. Clearly, further investigation is necessary to elucidate the precise pathway involved in this arrest, and to determine whether there is a checkpoint that monitors kinetochore assembly before entry into mitosis (Blower, 2001).

Genetic and protein localization studies have implicated several proteins in regulating centromere function. Until now, it has been difficult to determine whether these proteins are involved in kinetochore formation and function, other centromere functions, or functions independent of the centromere/kinetochore. The structural and functional analyses presented in this study support the hypothesis that distinct spatial and functional domains exist in the centromere and adjacent regions. Previously, cytological studies in humans have revealed the presence of spatially distinct protein domains in the mitotic kinetochore (Blower, 2001).

This study extends these observations by investigating the domain structure of the kinetochore and the centromere region, including the flanking heterochromatin, and has provided data that reveal the functional separation and interdependence of these structural domains. Centromeric chromatin is the central and most essential component of the centromere region, and is required for entry into mitosis and chromosome movement during mitosis, as well as for recruiting proteins to the kinetochore and flanking domains. The domain organization of the centromere observed in Drosophila is similar to the situation in S. pombe, where different proteins occupy distinct subdomains within the centromere region, and are required for separable chromosome segregation processes (Blower, 2001).

Studies in a variety of organisms have indicated that the centromere region is a site of specialized sister cohesion, not only in meiosis I but also in mitosis. For example, normal homolog disjunction requires that Mei-S332 functions to maintain sister chromatid cohesion in the centric regions throughout meiosis I and until anaphase of meiosis II. Although Mei-S332 is involved in sister chromatid cohesion, it is not a cohesin; moreover, its localization differs from that observed in S. cerevisiae, where cohesins are concentrated at the centromere and associated with centromeric chromatin (Blower, 2001).

Mei-S332 is required for proper chromosome inheritance in Drosophila, but surprisingly the chromosome still retains kinetochore protein localization and functions if Mei-S332 is eliminated. Despite the spatial and functional separation of Mei-S332 and Cid, Mei-S332 is dependent on functional centromeric chromatin for its recruitment to the centromere region: it is mislocalized in anti-Cid injected embryos and RNAi-treated Kc cells. Cid localization is not, however, dependent on the presence of Mei-S332. Therefore, Cid is epistatic to Mei-S332 in the pathway responsible for the assembly and/or maintenance of this physically and functionally distinct centromere region domain in mitosis. The relationship between Cid, kinetochore function and Mei-S332-mediated cohesion warrants further genetic and biochemical analyses. It will be particularly interesting to determine the significance of the consistent asymmetric positioning of Mei-S332 to only one side of the Cid chromatin, as well as its impact on Cid during meiosis, where mutant phenotypes are more severe (Blower, 2001).

Heterochromatin encodes several inheritance functions, including homolog pairing in meiosis I, sister chromatid cohesion and interactions with anti-poleward forces. The conserved location of centromeres in heterochromatin suggests that heterochromatin proteins, such as Prod and HP1, may be required for establishing or maintaining centromeres. Neither Prod nor HP1 are detectable in Cid chromatin and prod and Su(var)2-5 mutations do not affect the localization of Cid. Furthermore, neither Prod nor HP1 localization is affected in anti-Cid-injected embryos or RNAi-treated Kc cells (Blower, 2001).

It is concluded that Prod and HP1 function in the pericentromeric regions to promote normal condensation and chromosome segregation -- processes distinct from the centromere/kinetochore. Although the kinetochore is typically embedded in large blocks of heterochromatin, evidence has been provideed that it may be structurally and functionally distinct from the closely juxtaposed pericentromeric or centric heterochromatin (Blower, 2001).

If centromere chromatin structure is distinct from centric heterochromatin, why are centromeres embedded in heterochromatin in almost all multicellular eukaryotes? Perhaps the flanking heterochromatin does provide an environment that is necessary for the formation of a centromere-specific higher order chromatin structure. In addition, although Prod and HP1 are not necessary for Cid localization, they may encode functions redundant with other heterochromatic proteins that establish or maintain proper kinetochore structure. It will be interesting to determine whether protein localization and mutant analyses with other centric heterochromatin proteins are consistent with the results for prod and Su(var)2-5 (Blower, 2001).

Drosophila CENP-A/Cid is required for kinetochore formation and mitotic function, cell-cycle progression and recruiting transient kinetochore components and a sister chromatid cohesion protein. In contrast, Cid and proteins that function in the pericentric heterochromatin are physically and functionally independent. These results support the hypothesis that CENP-A proteins are central to many mitotic processes, and may be a component of the epigenetic mark responsible for centromere identity and function. Future studies should investigate what mechanism is responsible for loading CENP-A specifically into CENP-A chromatin in a replication-independent manner, since this process may be the key to understanding maintenance of the epigenetic mark and centromere identity (Blower, 2001).

Centromeric nucleosomes induce positive DNA supercoils

Centromeres of higher eukaryotes are epigenetically maintained; however, the mechanism that underlies centromere inheritance is unknown. Centromere identity and inheritance require the assembly of nucleosomes containing the CenH3 histone variant (Centromere identifier) in place of canonical H3. Although H3 nucleosomes wrap DNA in a left-handed manner and induce negative supercoils, this study shows that CenH3 nucleosomes reconstituted from Drosophila histones induce positive supercoils. Furthermore, CenH3 likewise induces positive supercoils in functional centromeres in vivo, using a budding yeast minichromosome system and temperature-sensitive mutations in kinetochore proteins. The right-handed wrapping of DNA around the histone core implied by positive supercoiling indicates that centromere nucleosomes are unlikely to be octameric and that the exposed surfaces holding the nucleosome together would be available for kinetochore protein recruitment. The mutual incompatibility of nucleosomes with opposite topologies could explain how centromeres are efficiently maintained as unique loci on chromosomes (Furuyama, 2009).

CenH3 nucleosomes induce positive supercoils, both when D. melanogaster CID is reconstituted into nucleosomes in vitro, and when S. cerevisiae Cse4p is assembled at functional minichromosome centromeres in vivo. This behavior is in stark contrast to canonical nucleosomes, in which the left-handed wrapping leads to induction of negative supercoils in topological assays. These observations of positive supercoiling induced by CenH3 from eukaryotic taxa as different as animals and fungi can be explained by either of two general models: overtwisting with left-handed wrapping or right-handed wrapping (Furuyama, 2009).

In a covalently closed circle, overtwisting of DNA (positive ΔTw) causes compensatory negative writhe that is removed by topoisomerase, resulting in a net positive ΔLk after deproteination. If CenH3 nucleosomes are left-handed octamers (Wr = −1), ΔTw would need to be +2 in order to result in a ΔLk of +1 (ΔLk = ΔTw + ΔW). Although the reported value of ΔWr for left-handed octamers varies, this study used the most conservative cited value of −1 to calculate the degree of overtwisting consistent with left-handed wrapping. The change required in the helical periodicity of DNA (Δh) to gain ΔTw = +2 and cancel one negative writhe induced by a left-handed nucleosome can be calculated as Δh = −h2xΔTw/N, where h = N/Tw, where N is the number of base pairs wrapped around the nucleosome. If an octameric CenH3 nucleosome (N=150 bp) is assumed, Δh equals −1.47 for ΔTw = +2. This corresponds to a helical periodicity of 9.03 bp/turn (whereas h = 10.5 bp/turn for B-DNA free in solution). The situation is even more extreme for CenH3 hemisomes, which wrap 80-120 bp of DNA, because the same amount of twist must be taken up by the shorter span of DNA (helical periodicity of 7.74-8.66 bp/turn). These estimated values for helical twist are conservative in that they assume that the extra twist is distributed over the whole nucleosome, including the DNA that wraps H2A/H2B dimers, whereas in the crystal structure of the H3 nucleosome core particle the twist of DNA wrapping H2A/H2B is similar to that in free solution. In addition, DNaseI digestion of CID chromatin assembled in vitro resulted in a normal helical periodicity estimate of ~10 bp/turn, and electron microscopy of CID chromatin revealed a beads-on-a-string appearance, suggesting entry/exit crossing. Thus, existing data are inconsistent with positive ΔTw being the reason for the observed positive supercoiling (Furuyama, 2009).

The implausibility of such strongly overtwisted DNA wrapping around a left-handed nucleosome leads to the conclusion that positive supercoiling instead indicates a right-handed wrap. A right-handed nucleosome would satisfy the observed positive supercoiling of approximately one supercoil per CenH3 nucleosome without a significant change in B-DNA periodicity. Tetrameric archaeal nucleosomes also wrap DNA in a right-handed configuration, with a helical periodicity of 10-11bp/turn. Also, in the absence of H2A/H2B dimers, (H3/H4)2 tetramers are capable of spontaneously shifting between both left- and right-handed configurations, presumably without significant changes in helical twist (Furuyama, 2009).

Histone octamers capable of wrapping DNA into a right-handed configuration have never been observed. Because H3/H4 tetramers can wrap DNA in either direction, it is the creation of a left-handed ramp by addition of two H2A/H2B dimers that is incompatible with the right-handed structure. The crystal structure of the H3 nucleosome (H2A'-H2B'-H4'-H3'-H3-H4-H2B-H2A plus DNA) reveals that the N-terminal helix of H3, as well as the C-terminus of H4, contact the C-terminal docking domain of H2A', which are essential interactions that hold the octamer together. In addition, the interaction between H2A and H2A' within the octamer through their Loop 1 regions hold together the two gyres of the DNA superhelix (Luger, 1997). These interactions that hold the octamer together are expected to be disrupted in a right-handed nucleosome because they would face away from each other in the right-handed structure; therefore, there is a strong structural basis for the absence of right-handed octameric nucleosomes in eukaryotes. Without altering the twist of DNA significantly, the only structures that yield ΔLk = +1 other than a right-handed octamer are right-handed hemisomes with right entry/exit crossing, and left-handed hemisomes with right entry/exit crossing. A single superhelical turn of DNA around a hemisome results in a closer physical distance between the entry/exit DNA compared to that in an octameric structure, which has an additional turn between the two entry/exit sites (see Structures and model of a right-handed hemisome). Therefore, it is structurally very difficult to make a left-handed hemisome with a right-handed crossing (Furuyama, 2009).

In budding yeast, various model of Cse4p nucleosomes have been suggested, including octamers (H2A/H2B/H4/Cse4p/Cse4p/H4/H2B/H2A), hemisomes (Cse4p/H4/H2B/H2A) and nucleosomes containing the non-histone Scm3 protein substituting for H2A/H2B dimers (H4/Cse4p/Cse4p/H4)(Scm3)1-2. Given the current finding that Cse4p nucleosomes induce positive supercoils, it is unlikely that they can exist as octamers. Furthermore, the observation that Scm3 binds to the region of Cse4p required for the 4-helix bundle homodimerization interface of the octameric particle would a priori argue against a stable octameric particle. That leaves either Cse4p/H4/H2A/H2B hemisomes or Cse4p/H4/Scm3 particles as candidate yeast CenH3 nucleosomes. Both of these models are consistent with the localization of Cse4p to a small ~80bp CDEII region of CDE. It is attractive to suggest that right-handed hemisomes are conserved in all eukaryotes, because Cse4p can functionally replace human CENP-A (Furuyama, 2009).

There are several structural implications of right-handed hemisomes. The strong H3/H3 4-helix bundle at the dyad axis and the weak H4/H2B 4-helix bundles linking the central tetramer to flanking dimers precludes formation of H3/H4/H2B/H2A hemisomes, and indeed no stable H3 hemisomes have been observed. Therefore, the existence of CenH3 hemisomes suggests that CenH3 induces structural alterations that stabilize the tetrameric particle. The crossing of entry/exit DNA in the CenH3 hemisome may be an important feature, because it can potentially stabilize the hemisome. In contrast, the entry/exit DNA of H3 octameric nucleosomes does not cross most of the time, but rather is occupied by a linker histone. Consistent with this difference, the H1 linker histone is depleted from centromeric chromatin, and the H5 linker histone is incapable of associating with human CENP-A nucleosomes in vitro. In addition, surfaces involved in contacts within left-handed octameric nucleosomes will be exposed in right-handed hemisomes, such as the C-terminal docking domain of H2A and the N-terminal helix of H3. A right-handed configuration also changes the relative position of these domains. The combination of additional exposed surfaces and altered presentation of the same surfaces might provide essential interaction domains for kinetochore proteins to assemble functional centromeres (Furuyama, 2009).

The finding that CenH3 nucleosomes are right-handed also might help explain why key residues involved in H3/H3 4-helix bundle formation are invariant in CenH3s, despite considerable divergence elsewhere in the core. This observation suggests that the CenH3 dimerization interface is occupied under at least some circumstances. It is suggested that this interface has been retained to permit CenH3/H3 hybrid formation, which would result in left/right core particles that should be unable to stably wrap DNA. Misincorporation of CenH3 outside of centromeres occurs under many circumstances, yet is potentially catastrophic, causing dicentric formation, chromosome loss and dominant lethality. By retaining the ability to dimerize with H3, misincorporated CenH3s would predominantly form structurally defective nucleosomes, thus helping to maintain the extraordinary fidelity of centromere maintenance (Furuyama, 2009).

At the boundary between CenH3 and H3 nucleosomal arrays, the change in the direction of DNA around histones from left-handed to right-handed might also have profound implications for maintaining functional centromeres. The uniform packaging of H3 nucleosomes in pericentric heterochromatin, induced in part by the uniform size of centromeric satellite repeats, is expected to be disturbed by the sudden change in the direction of DNA wrapping around CenH3. This would result in a higher-order structural transition from near-crystalline rigid heterochromatin to less densely packaged centromeric chromatin as implied by the unusually long linker DNA found in Drosophila centromeric chromatin. The octameric form of canonical H3 nucleosomes is believed to represent a critical evolutionary leap in being able to more densely package the genome, whereas tetrameric archael nucleosomes fail to condense into a comparable higher order packaging. Therefore, the presence of a CenH3 hemisome array that packages DNA in a right-handed orientation and resists octameric packaging would provide a singular location that remains decondensed during mitosis and accessible to binding by kinetochore proteins. The mutual incompatibility of nucleosome cores that wrap DNA in opposite directions suggests a novel mechanism for perpetual maintenance of the centromere within a chromosomal landscape that is dominated by conventional chromatin (Furuyama, 2009).

The histone H3 variant CENP-A epigenetically defines the centromere and is critical for chromosome segregation. This study reports an interaction between CENP-A and subunits of the mitochondrial ATP synthase complex in the germline of male Drosophila. Furthermore, knockdown of CENP-A, as well as subunits ATPsyn-alpha, ATPsyn-betalike (a testis-specific paralogue of ATPsyn-beta) and ATPsyn-gamma disrupts sister centromere cohesion in meiotic prophase I. This disruption is likely independent of reduced ATP levels. ATPsyn-alpha and -betalike were found to localise to meiotic centromeres; and this localisation is dependent on the presence of CENP-A. ATPsyn-alpha directly interacts with the N-terminus of CENP-A in vitro, and truncation of its N terminus perturbs sister centromere cohesion in prophase I. It is proposed that the CENP-A N-terminus recruits ATPsyn-alpha and -betalike to centromeres to promote sister centromere cohesion in a nuclear function that is independent of oxidative phosphorylation (Collins, 2018).

Meiosis is the specialised cell division cycle in which one round of DNA replication precedes two rounds of chromosome segregation that generate haploid gametes (eggs and sperm). Defects in meiosis lead to reduced fertility, sterility or aneuploidy in gametes or resulting zygotes. Centromeres, defined epigenetically by incorporation of the histone H3 variant CENP-A2 play a key role in coordinating meiotic chromosome segregation. Studies in plants suggest that CENP-A adopts meiosis-specific functions via its highly divergent N terminus. To investigate functions of the CENP-A N terminus in meiosis in an animal, this study used biochemical and genetic approaches in testis of Drosophila males. Unexpected functional links were uncovered between CENP-A, mitochondrial ATP synthase F1 subunits and sister centromere cohesion in meiosis. It is proposed that the CENP-A N-terminus recruits ATPsyn-α and -βlike, a testis-specific paralogue of -β, to centromeres to promote sister centromere cohesion in a novel nuclear function that is independent of canonical roles in oxidative phosphorylation (Collins, 2018).

In addition to an expected function in ATP synthesis, this paper reports a function for ATPsyn-α and ATPsyn-βlike in male meiosis and fertility. In testes depleted for ATPsyn-α or –βlike, prophase I cells accumulate prior to meiosis I, providing a possible explanation for observed sterility in previously isolated mutants. Given that canonical ATPsyn-β expression in testis is reduced compared to whole adults, ATPsyn-βlike might normally compensate for ATPsyn-β function. Moreover, although the expression pattern of ATPsyn-βlike is entirely consistent with a testis-specific function, ATPsyn-βlike expression is noted at larval and pupal stages (modENCODE RNA-Seq). This raises the possibility that ATPsyn-βlike adopts additional functions in development, which were not addressed in this study. In addition to its canonical role, a nuclear function is reported for ATPsyn-α and -βlike, in particular at centromeres. ATPsyn-α and -βlike localise to centromeres at meiotic prophase I, and this localisation requires CENP-A. Moreover, CENP-A, ATPsyn-α and ATPsyn-βlike are each required to maintain sister centromere cohesion at this stage. Remarkably, ATPsyn-βlike specifies the enrichment of the cohesion protector MEI-S322 to centromeres at prometaphase I, perhaps comparable to the Chromosome Passenger Complex subunit INCENP. In contrast, ATPsyn-α appears to have a distinct function in the nuclear and centromeric localisation of MEI-S332. MEI-S332 mislocalisation to global chromatin in ATPsyn-βlike-depleted nuclei is particularly striking and might be a consequence of a sustained prometaphase I arrest or indicates a more general function of ATPsyn-βlike on chromatin. In Drosophila males, an alternative cohesin complex made up of ORD, SOLO and SUNN maintains meiotic sister centromere cohesion at late prophase I S6. This study also found that CENP-A is required for centromere cohesion early in prophase I at S1/2a, prior to ORD, SOLO and SUNN. It is suggested that observed defects in cohesion lead to failed progression through meiosis I and ultimately reduced fertility or sterility. Intriguingly, depletion of ATP synthase F1 subunits also disrupts sister chromatid arm cohesion and 4th homologue pairing/cohesion, suggesting additional global functions outside of the centromere. The ATPsyn-α subunit directly interacts with the CENP-A N terminus, providing a first function for conserved B1 and B2 domains. It is proposed that ATPsyn-α recruits ATPsyn-βlike to centromeres. This functional analyses of flies expressing a GFP-tagged CENP-A lacking amino acids 1–118 show the CENP-A N terminus is not required for meiotic centromere localisation, different from plants. Instead, the fly CENP-A N terminus appears to be important for meiotic sister centromere cohesion, possibly via the recruitment of ATPsyn-α and ATPsyn-βlike (Collins, 2018).

These data support a model in which mitochondrial ATP synthase F1 subunits adopt nuclear functions that appear to be independent of ATP production. First, ATPsyn-α and ATPsyn-βlike interact with CENP-A/centromeres. Second, the severity of observed meiotic phenotypes does not correlate with ATP supply. Third, ATP depletion was not sufficient to induce a loss of cohesion. Finally, these findings are in line with an ATP independent requirement for ATPsyn-α, -β and -γ in germ line stem cell differentiation in Drosophila females. In conclusion, it is proposed that the CENP-A N-terminus recruits ATPsyn-α and ATPsyn-βlike to centromeres to promote sister centromere cohesion in a novel nuclear function that is independent of canonical roles in oxidative phosphorylation (Collins, 2018).

PROTEIN STRUCTURE

Amino Acids - 225

Structural Domains

Mammalia CENP-A and yeast Cse4p share with histone H3 the ~100-aa
nucleosomal core, but have N-terminal tails that are completely dissimilar from one another and from H3. In CENP-A,
centromeric localization maps to the core, which is about equally divergent from both Cse4p and H3. Except for
CENP-A and Cse4p, only three GenBank entries were found to have comparably diverged H3-like cores and dissimilar N-terminal tails. A 225-aa ORF that fits this profile has been found within a 'sequencing in progress' database entry (AC005652) deposited by the Berkeley Drosophila Genome Project.
The possibility was considered that this ORF encodes a centromeric protein. An antiserum against a
peptide predicted from the ORF was used for immunocytochemistry to D. melanogaster Kc cells. Intense, point-like signals were observed over interphase nuclei
and exclusively over centromeric constrictions of all Drosophila mitotic chromosomes in both Kc tissue culture cells and larval neuroblasts. Similar
results were obtained with antibody raised against a peptide predicted from another N-terminal tail region of the ORF. Therefore, these epitopes
are present on a normal centromere component that is present throughout the cell cycle. Because the 225-aa protein behaves as a Drosophila homolog of CENP-A and Cse4p, the gene was named cid (for centromere identifier) (Henikoff, 2000).